Detecting regions of homozygosity improves the diagnosis of pathogenic variants and uniparental disomy in pediatric patients.

Chromosomal microarray analysis using single nucleotide polymorphism probes can detect regions of homozygosity (ROH). This confers a potential utility in revealing autosomal recessive (AR) diseases and uniparental disomy (UPD). Results of genetic testing among pediatric patients from 2015 to 2019 were evaluated. Diagnostic findings with detected ROH from large consecutive case series in the literature were reviewed. Of 2050 pediatric patients, 65 (3%) had one or more ROH and 31 (53%) had follow-up whole exome sequencing (WES) and methylation studies. Seven homozygous variants were detected and four of them from three patients (9.6%) were within the detected ROH and classified as pathogenic or likely pathogenic variants for AR diseases. One patient (3%) had segmental UPD15q for a diagnosis of Prader-Willi syndrome. Additive diagnostic yield from ROH reporting was at least 0.2% (4/2050) of pediatric patients. These results were consistent with findings from several large case series reported in the literature. Detecting ROH had an estimated baseline predictive value of 10% for AR diseases and 3% for UPD. Consanguinity revealed by multiple ROH was a strong predictor for AR diseases. These results provide evidence for genetic counseling and recommendation of follow-up WES and methylation studies for pediatric patients reported with ROH.

Ring chromosome formation by intra-strand repairing of subtelomeric double stand breaks and clinico-cytogenomic correlations for ring chromosome 9.

Constitutional ring chromosome 9, r(9), is a rare chromosomal disorder. Cytogenomic analyses by karyotyping, array comparative genomic hybridization (aCGH) and whole genome sequencing (WGS) were performed in a patient of r(9). Karyotyping detected a mosaic pattern of r(9) and monosomy 9 in 83% and 17% of cells, respectively. aCGH detected subtelomeric deletions of 407 kb at 9p24.3 and 884 kb at 9q34.3 and an interstitial duplication of 5.879 Mb at 9q33.2q34.11. WGS revealed double strand breaks (DSBs) at ends of 9p24.3 and 9q34.3, inverted repeats at ends of subtelomeric and 9q33.2q34.11 regions, and microhomology sequences at the junctions of this r(9). This is the first report of r(9) analyzed by WGS to delineate the mechanism of ring chromosome formation from repairing of subtelomeric DSBs. The loss of telomeres by subtelomeric DSBs triggered inverted repeats induced intra-strand foldback and then microhomology mediated synthesis and ligation, which resulted in the formation of this r(9) with distal deletions and an interstitial duplication. Review of literature found seven patients of r(9) with clinical and cytogenomic findings. These patients and the present patient were registered into the Human Ring Chromosome Registry and a map correlating critical regions and candidate genes with relevant phenotypes was constructed. Variable phenotypes of r(9) patients could be explained by critical regions and genes of DOCK8, DMRT, SMARCA2, CD274, IL33, PTPRD, CER1, FREM1 for 9p deletions, and the EHMT1 gene for 9q34 deletion syndrome. This interactive registry of r(9) could provide information for cytogenomic diagnosis, genetics counseling and clinical management.

Genotype-Phenotype Correlations for Putative Haploinsufficient Genes in Deletions of 6q26-q27: Report of Eight Patients and Review of Literature.

Background Cytogenomic analyses have been used to detect pathogenic copy number variants. Patients with deletions at 6q26-q27 present variable clinical features. We reported clinical and cytogenomic findings of eight unrelated patients with a deletion of 6q26-q27. A systematic review of the literature found 28 patients with a deletion of 6q26-q27 from 2010 to 2020. Results For these 36 patients, the sex ratio showed equal occurrence between males and females; 29 patients (81%) had a terminal deletion and seven patients (19%) had a proximal or distal interstitial deletion. Of the 22 patients with parental studies, deletions of de novo, maternal, paternal, and bi-parental inheritance accounted for 64, 18, 14, and 4% of patients, respectively. The most common clinical findings were brain abnormalities (100%) in fetuses observed by ultrasonography followed by developmental delay and intellectual disability (81%), brain abnormalities (72%), facial dysmorphism (66%), hypotonia (63%), learning difficulty or language delay (50%), and seizures (47%) in pediatric and adult patients. Anti-epilepsy treatment showed the effect on controlling seizures in these patients. Cytogenomic mapping defined one proximal critical region at 6q26 containing the putative haploinsufficient gene PRKN and one distal critical region at 6q27 containing two haploinsufficient genes DLL1 and TBP . Deletions involving the PRKN gene could associate with early-onset Parkinson disease and autism spectrum disorder; deletions involving the DLL1 gene correlate with the 6q terminal deletion syndrome. Conclusion The genotype-phenotype correlations for putative haploinsufficient genes in deletions of 6q26-q27 provided evidence for precise diagnostic interpretation, genetic counseling, and clinical management of patients with a deletion of 6q26-q27.

Cytogenomic Characterization of Giant Ring or Rod Marker Chromosome in Four Cases of Well-Differentiated and Dedifferentiated Liposarcoma.

Chromosome and array comparative genomic hybridization (aCGH) analyses were performed on two cases of well-differentiated liposarcoma (WDLPS) and two cases of dedifferentiated liposarcoma (DDLPS). The results revealed the characteristic giant ring (GR) or giant rod marker (GRM) chromosomes in all four cases and amplification of numerous somatic copy number alterations (SCNAs) involving a core segment of 12q14.1q15 and other chromosomal regions in three cases. The levels of amplification for oncogenes OS9, CDK4, HMGA2, NUP107, MDM2, YEATS4, and FRS2 at the core segment or other SCNAs should be characterized to facilitate pathologic correlation and prognostic prediction. Further studies for the initial cellular crisis event affecting chromosome intermingling regions for cell-type specific gene regulation may reveal the underlying mutagenesis mechanism for GR and GRM in WDLPS and DDLPS.

Detection of cytogenomic abnormalities by OncoScan microarray assay for products of conception from formalin-fixed paraffin-embedded and fresh fetal tissues.

BACKGROUND: The OncoScan microarray assay (OMA) using highly multiplexed molecular inversion probes for single nucleotide polymorphism (SNP) loci enabled the detection of cytogenomic abnormalities of chromosomal imbalances and pathogenic copy number variants (pCNV). The small size of molecular inversion probes is optimal for SNP genotyping of fragmented DNA from fixed tissues. This retrospective study evaluated the clinical utility of OMA as a uniform platform to detect cytogenomic abnormalities for pregnancy loss from fresh and fixed tissues of products of conception (POC). RESULTS: Fresh specimens of POC were routinely subjected to cell culture and then analyzed by karyotyping. POC specimens with a normal karyotype (NK) or culture failure (CF) and from formalin-fixed paraffin-embedded (FFPE) tissues were subjected to DNA extraction for OMA. The abnormality detection rate (ADR) by OMA on 94 cases of POC-NK, 38 cases of POC-CF, and 35 cases of POC-FFPE tissues were 2% (2/94), 26% (10/38), and 57% (20/35), respectively. The detected cytogenomic abnormalities of aneuploidies, triploidies and pCNV accounted for 50%, 40% and 10% in POC-CF and 85%, 10% and 5% in POC-FFPE, respectively. False negative result from cultured maternal cells and maternal cell contamination were each detected in one case. OMA on two cases with unbalanced structural chromosome abnormalities further defined genomic imbalances and breakpoints. CONCLUSION: OMA on POC-CF and POC-FFPE showed a high diagnostic yield of cytogenomic abnormalities. This approach circumvented the obstacles of CF from fresh specimens and fragmented DNA from fixed tissues and provided a reliable and effective platform for detecting cytogenomic abnormalities and monitoring true fetal result from maternal cell contamination.

Cytogenomic Abnormalities in 19 Cases of Salivary Gland Tumors of Parotid Gland Origin.

Salivary gland tumors (SGTs) of parotid origin are a group of diverse neoplasms which are difficult to classify due to their rarity and similar morphologic patterns. Chromosome analysis can detect clonal abnormalities, and array comparative genomic hybridization (aCGH) analysis can define copy number alterations (CNAs) from tumor specimens. Of the 19 cases of various types of SGTs submitted for cytogenomic analyses, an abnormal clone was detected in nine cases (47%), and CNAs were detected in 14 cases (74%). Recurrent rearrangements involving the PLAG1 gene at 8q12, recurrent CNAs including deletions of 6q, 9p (CDKN2A), and 17p (TP53), loss of Y chromosome, and gain of chromosome 7 were defined from these cases. Combined karyotyping and aCGH analyses could improve diagnostic yield. Future study for more precisive correlation of SGT classification with cytogenomic abnormalities will facilitate better diagnosis and treatment.

Diagnostic cytogenetic testing following positive noninvasive prenatal screening results of sex chromosome abnormalities: Report of five cases and systematic review of evidence.

BACKGROUND: Follow-up cytogenetic analysis has been recommended for cases with positive noninvasive prenatal screening (NIPS) results. This study of five cases with numerical and structural sex chromosomal abnormalities (SCA) and a review of large case series of NIPS provided guidance to improve prenatal diagnosis for SCA. METHODS: Following positive NIPS results for SCA, karyotype analysis, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH), and locus-specific quantitative PCR were performed on cultured amniocytes, chorionic villi cells, and stimulated lymphocytes. Review of large case series was performed to evaluate the NIPS positive rate, follow-up rate of cytogenetic analysis, positive predictive value (PPV) for major types of SCA, and relative frequencies of subtypes of major SCA. RESULTS: Of the five cases with positive NIPS for SCA, case 1 showed a mosaic pattern of monosomy X and isodicentric Y; case 2 showed a mosaic pattern of monosomy X confined to the placenta; cases 3 and 4 had an isochromosome of Xq, and case 5 showed a derivative chromosome 14 from a Yq/14p translocation of maternal origin. Review of literature showed that mean positive rate of NIPS for SCA was 0.61%, follow-up rate of cytogenetics analysis was 76%, and mean PPV for SCA was 48%. Mosaic patterns and structural rearrangements involving sex chromosomes were estimated in 3%-20% and 3% of SCA cases, respectively. CONCLUSION: These five cases further demonstrated the necessity to pursue follow-up cytogenetic analysis to characterize mosaic patterns and structural abnormalities involving sex chromosomes and their value for prenatal genetic counseling. A workflow showing the performance of current NIPS and cytogenetic analysis for SCA was summarized. These results could facilitate an evidence-based approach to guide prenatal diagnosis of SCA.

Inverted duplication, triplication and quintuplication through sequential breakage-fusion-bridge events induced by a terminal deletion at 5p in a case of spontaneous abortion.

BACKGROUND: Integrated chromosome, fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) analyses have been effective in defining unbalanced chromosomal rearrangements. Discordant chromosome and aCGH results are rarely reported. METHODS: Routine cytogenomic analyses and literature review were performed in the study of a case from products of conception (POC). RESULTS: Chromosome and FISH analysis revealed a mosaic pattern consisting of a primary aberration of an inverted duplication of 5p and derived secondary and tertiary aberrations from sequential triplication and quintuplication of 5p, respectively. The aCGH analysis detected only a 1.521 Mb terminal deletion at 5p15.33 with no other pathogenic copy number variants in the genome. This mosaic karyotypic pattern likely resulted from chromosome instability induced by sequential breakage-fusion-bridge events during in vitro cell culture. A review of literature found heterogeneous distal deletion and inverted duplication of 5p in prenatal and pediatric cases. CONCLUSION: This is the first case reported in POC with a unique mosaic pattern and discordant chromosome and aCGH results. Caution should be applied in reporting and interpreting these discordant results and further analysis for underlying mechanism should be considered.

Integrated FISH, Karyotyping and aCGH Analyses for Effective Prenatal Diagnosis of Common Aneuploidies and Other Cytogenomic Abnormalities.

Current prenatal genetic evaluation showed a significantly increase in non-invasive screening and the reduction of invasive diagnostic procedures. To evaluate the diagnostic efficacy on detecting common aneuploidies, structural chromosomal rearrangements, and pathogenic copy number variants (pCNV), we performed a retrospective analysis on a case series initially analyzed by aneuvysion fluorescence in situ hybridization (FISH) and karyotyping then followed by array comparative genomic hybridization (aCGH). Of the 386 cases retrieved from the past decade, common aneuploidies were detected in 137 cases (35.5%), other chromosomal structural rearrangements were detected in four cases (1%), and pCNV were detected in five cases (1.3%). The relative frequencies for common aneuploidies suggested an under detection of sex chromosome aneuploidies. Approximately 9.5% of cases with common aneuploidies showed a mosaic pattern. Inconsistent results between FISH and karyotyping were noted in cases with pseudo-mosaicism introduced by culture artifact or variable cellular proliferation from cells with mosaic karyotypic complements under in vitro cell culture. Based on findings from this case series, cell-based FISH and karyotyping should be performed to detect common aneuploidies, structural chromosomal abnormalities, and mosaic pattern. DNA-based aCGH and reflex FISH should be performed to detect and confirm genomic imbalances and pCNV. Practice points to ensure the diagnostic accuracy and efficacy were summarized.

Analytical validation and chromosomal distribution of regions of homozygosity by oligonucleotide array comparative genomic hybridization from normal prenatal and postnatal case series.

BACKGROUND: Regions of homozygosity (ROH) are continuous homozygous segments commonly seen in the human genome. The integration of single nucleotide polymorphism (SNP) probes into current array comparative genomic hybridization (aCGH) analysis has enabled the detection of the ROH. However, for detecting and reporting biologically relevant ROH in a clinical setting, it is necessary to assess the analytical validity of SNP calling and the chromosomal distribution of ROH in normal populations. METHODS: The analytical validity was evaluated by correlating the consistency of SNP calling with the quality parameters of aCGH and by accessing the accuracy of SNP calling using PCR based restriction enzyme digestion and Sanger sequencing. The distribution of ROH was evaluated by the numbers, sizes, locations, and frequencies of ROH from the collection of data from parental, postnatal, and prenatal case series that had normal aCGH and chromosome results. RESULTS: The SNP calling failure rate was 20-30% with a derivative Log2 ratio (DLR) below 0.2 and increased significantly to 30-40% with DLR of 0.2-0.4. The accuracy of SNP calling is 93%. Of the 958 cases tested, 34% had no ROH, 64% had one to four ROH, and less than 1% had more than five ROH. Of the 1196 ROH detected, 95% were less than 10 Mb. The distribution of numbers and sizes of ROH showed no differences among the parental, pediatric and prenatal case series and test tissues. The chromosomal distribution of ROH was non-random with ROH seen most frequently in chromosome 8, less frequently in chromosomes 2, 6, 10, 12, 11 and 18, and most rarely seen on chromosomes 15, 19, 21 and 22. Recurrent ROH occurring with a frequency greater than 1% were detected in 17 chromosomal loci which locates either in the pericentric or interstitial regions. CONCLUSION: With a quality control parameter of DLR set at below 0.2, the consistency of SNP calling would be 75%, the accuracy of SNP call could be 93%, and the observed chromosomal distribution of ROH could be used as a reference. This aCGH analysis could be a reliable screening tool to document biologically relevant ROH and recommend further molecular analysis.

A Retrospective Analysis of 10-Year Data Assessed the Diagnostic Accuracy and Efficacy of Cytogenomic Abnormalities in Current Prenatal and Pediatric Settings.

Background: Array comparative genomic hybridization (aCGH), karyotyping and fluorescence in situ hybridization (FISH) analyses have been used in a clinical cytogenetic laboratory. A systematic analysis on diagnostic findings of cytogenomic abnormalities in current prenatal and pediatric settings provides approaches for future improvement. Methods: A retrospective analysis was performed on abnormal findings by aCGH, karyotyping, and FISH from 3,608 prenatal cases and 4,509 pediatric cases during 2008-2017. The diagnostic accuracy was evaluated by comparing the abnormality detection rate (ADR) and the relative frequency (RF) of different types of cytogenomic abnormalities between prenatal and pediatric cases. A linear regression correlation between known prevalence and ADR of genomic disorders was used to extrapolate the prevalence of other genomic disorders. The diagnostic efficacy was estimated as percentage of detected abnormal cases by expected abnormal cases from served population. Results: The composite ADR for numerical chromosome abnormalities, structural chromosome abnormalities, recurrent genomic disorders, and sporadic pathogenic copy number variants (pCNVs) in prenatal cases were 13.03%, 1.77%, 1.69%, and 0.9%, respectively, and were 5.13%, 2.84%, 7.08%, and 2.69% in pediatric cases, respectively. The chromosomal abnormalities detected in prenatal cases (14.80%) were significantly higher than that of pediatric cases (7.97%) (p < 0.05), while the pCNVs detected in prenatal cases (2.59%) were significantly lower than that of pediatric cases (9.77%) (p < 0.05). The prevalence of recurrent genomic disorders and total pCNVs was estimated to be 1/396 and 1/291, respectively. Approximately, 29% and 35% of cytogenomic abnormalities expected from the population served were detected in current prenatal and pediatric diagnostic practice, respectively. Conclusion: For chromosomal abnormalities, effective detection of Down syndrome (DS) and Turner syndrome (TS) and under detection of sex chromosome numerical abnormalities in both prenatal and pediatric cases were noted. For pCNVs, under detection of pCNVs in prenatal cases and effective detection of DiGeorge syndrome (DGS) and variable efficacy in detecting other pCNVs in pediatric cases were noted. Extend aCGH analysis to more prenatal cases with fetal ultrasonographic anomalies, enhanced non-invasive prenatal (NIPT) testing screening for syndromic genomic disorders, and better clinical indications for pCNVs are approaches that could improve diagnostic yield of cytogenomic abnormalities.